The present invention relates to a method of encapsulating cationic species in liposomes and, more particularly, to a method of encapsulating high concentrations of cationic species in unilamellar and multilamellar vesicles.
Liposome vesicles are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilameller vesicles (onion-like structures characterized by two or more membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient towards the center of the bilayer while the hydrophilic "heads" orient towards the aqueous phase.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965, 12:238-252 involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638).
An ability to encapsulate high concentrations of divalent and trivalent cations in the aqueous compartment of a liposome, such as LUVs, has many potential applications. For example, encapsulation of gamma-emitting radionucleotides such as gadolinium (.sup.153 Gd.sup.3+), gallium (.sup.67 Ga.sup.3+), and indium (.sup.111 In.sup.3+) may be used for contrast enhanced nuclear magnetic resonance imaging and determining the biodistribution of liposomes in vivo, Turner et al., Radiology, 166, 761-765 (1988). Furthermore, the encapsulation of divalent and trivalent cations such gold (Au.sup.3+) and iron (Fe.sup.++) or barium (Ba.sup.++), in addition to uranium and lead, may be suitable for the preparation of electron dense or "heavy" LUVs which may have utility in electron microscopy studies or liposome/plasma separation procedures.
While the desirability of incorporating high concentrations of cations within the aqueous compartment of LUVs has been recognized, it has proven to be difficult in practice to achieve such high concentrations. In this regard, a number of processes have been proposed. For example, there has been proposed the use of passive entrapment to load the cation into the vesicle. However, such technique has been observed to result in low trapping efficiencies. Hwang et al., PNAS 74, 4991-4995 (1978). The use of lipophilic carboxylic ionophoretic antibiotics such as A23187 has been proposed for increasing the cation permeability of lipid membranes by forming a complex with cations. Reed et al., J. Biological Chemistry 247, 6970-6977 (1972). In effect, such technique increases the solubility of the cations in the lipid phase since the ionophore acts as a cation carrier. Nonetheless, the use of such ionophores as a method of remote cation loading in general results in an equilibrium distribution of the cation. This, of course, translates into entrapment of undesirably low levels of the extravesicular cation. Kolber et al., Biophysics Journal, 36, 369-391.
Techniques have also been developed for encapsulating cationic species into an LUV in amounts greater than achievable under equilibrium conditions. For example, the entrapment of appropriate chelating agents has been found to enable entrapment of higher levels of the cation. Nonetheless, such technique is limited to some extent by the limit in the interior concentration of the chelating agent which can be achieved, which is affected by the specificity of the chelator for the cation being loaded. In certain situations, binding affinities of the chelator have been found not to be stronger than that of the ionophore employed. Hwang, Journal of Nuclear Medicine, 19, 1162-70 (1978); Mauk et al., Analytical Biochemistry, 94, 302-307 (1979); Hwang et al., BBA, 716, 101-109 (1982).
Similar problems have been encountered with multilamellar vesicles (MLVs).
Methods have been disclosed for the entrapment of higher than equilibrium concentrations of ionizable bioactive agents, such as ionizable antineoplastic agents, when the vesicles employed possess a transmembrane concentration gradient (Bally et al., International Publication No. WO 86/01102, published Feb. 27, 1986, entitled "Encapsulation of Antineoplastic Agents in Liposomes"). Such a transmembrane gradient can be established via a sodium-potassium (Na.sup.+ /K.sup.+) or a pH gradient, gradients which reflect loading of monovalent cartons, K.sup.+ or H.sup.+. The gradient loads the ionizable drug into the vesicle. The vesicles may additionally but not necessarily contain an ionophore such as valinomycin which aids in the loading of K.sup.+ into the vesicles.
The mechanism of this valinomycin-driven loading of K.sup.+ is via an ion-for-ion exchange of Na.sup.+ for K.sup.+, or ion for H.sup.+ (in the case of a transmembrane pH gradient) creating an established equilibrium, unlike the mechanism of the instant ionophores. The instant invention loads cartons into vesicles as a result of a transmembrane pH gradient and an ionophore, both of which are required in the practice of the invention, but not as a result of an exchange mechanism.